HIV-1 Nef Assembles a Src Family Kinase-ZAP-70/Syk-PI3K Cascade to Downregulate Cell-Surface MHC-I

Abstract

HIV-1 Nef, which is required for the efficient onset of AIDS, enhances viral replication and infectivity by exerting multiple effects on infected cells. Nef downregulates cell-surface MHC-I molecules by an uncharacterized PI3K pathway requiring the actions of two Nef motifs-EEEE(65) and PXXP(75). We report that the Nef EEEE(65) targeting motif enables Nef PXXP(75) to bind and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K to trigger MHC-I downregulation in primary CD4+ T cells. In promonocytic cells, Nef/SFK recruits the ZAP-70 homolog Syk to downregulate MHC-I, implicating this PI3K pathway in multiple HIV-1 reservoirs. Isoform-specific PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agents to combat HIV-1. The discovery of this Nef-SFK-ZAP-70/Syk-PI3K signaling pathway explains the hierarchal role of the Nef motifs in effecting immunoevasion.

Protein–protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein

“…D1 cell treatment also reduced in a dose-dependent manner Nef-mediated MHC class I but not CD4 down-regulation (SI Fig. 7), which are, respectively, dependent and independent on the Nef-SH3 binding surface, as efficiently as the PI3K inhibitor LY294002 (13). The direct impact of D1 on Nef-SH3Hck interaction was then dose-dependently assessed in vitro by GST pull-down experiments.…”

“…induced pathogenicity and immune escape (13,17). Identification of these compounds was facilitated by a preliminary step of in silico screening that was next validated in a cell-based screening of the very same library of compounds, showing that a significant EF was attained in the preliminary virtual screening.…”

Caught in the act – protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease

“…This chink in the armor of HIV-1 provides an alternative approach to combat the virus by reversing the Nef-mediated immune evasion pathway. In support of this possibility, treatment of HIV-1-infected primary CD4 + T-cells with small-molecule inhibitors of the multi-kinase complex restores cell surface expression of MHC-I and sensitizes them to killing by CD8 + T-cells (Hung et al, 2007;Dikeakos et al, 2010;and M. Ostrowski, personal communication).…”

“…3B). To this end, Nef interacts with PACS-2 on early endosomes, which enables the HIV-1 protein to assemble a multi-kinase complex consisting of a Src family kinase (SFK), ζ-chain-associated protein kinase 70 (ZAP-70) and a class I phosphoinositide-3 kinase (PI3K) (Blagoveshchenskaya et al, 2002;Hung et al, 2007;Atkins et al, 2008). The activated multikinase complex increases the amount of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) underneath the plasma membrane, which recruits an ARF6 GEF to activate ARF6 and accelerate endocytosis of cell-surface MHC-I (Blagoveshchenskaya et al, 2002).…”

HIV-1 Nef Binds PACS-2 to Assemble a Multikinase Cascade That Triggers Major Histocompatibility Complex Class I (MHC-I) Down-regulation

“…Nef affects cells in many ways, including altering T-cell activation and maturation (3-5), subverting the apoptotic machinery, and down-regulating CD4 molecules and major histocompatibility complex class I (MHC-I) molecules encoded by the HLA-A and -B loci (2,6). But the precise mechanism of how Nef mediates these pathways has remained elusive.Nef diverts cell-surface MHC-I molecules to trans-Golgi network (TGN)-associated endosomal compartments by an endocytic pathway that is stimulated by class I phosphoinositide 3-kinase (PI3K) and dependent on ADP-ribosylation factor-6 (ARF6) (2,7,8). This MHC-I down-regulation requires the action of three motifs (1, 2) as follows: an N-proximal ␣-helical region (residues 7-26) (9) containing a critical methionine (Met 20 ) that promotes association of MHC-I with the heterotetrameric adaptor AP-1 (10, 11); an acidic cluster (EEEE 65 ) required for binding to phosphofurin acidic cluster sorting protein-1 (PACS-1) (12, 13); and an SH3-binding domain formed by a type II polyproline helix (PXXP 75 ) (9, 12) that promotes association of Nef with Src family tyrosine kinases (SFKs) (14 -17).…”